Measurement of material extravasation in microvascular networks using fluorescence video-microscopy

We have developed a new method using fluorescence videomicroscopy to quantitate the extravasation of intravenously injected materials. This method can measure the relative plasma concentration of, and the vascular permeability to, these materials in microcirculatory preparations which contain multiple blood vessels in a field of view. The image of a tissue area containing multiple blood vessels is recorded via a SIT camera immediately before, and for an extended period after, the intravenous injection of a bolus of fluorescent test tracers. The videotape is analyzed off-line. At various time points, the light intensities of the entire tissue area and of several spots over selected vessels are measured. These measurements are then used to calculate the fluorescent light intensities arising from the tracers inside vessels (Iv) and in the interstitial region (Ii). Iv represents the relative amount of the tracers in the plasma, and Ii represents that in the interstitium. Iv and Ii are used to calculate an average permeability (P) for the vessels in the observed region. The benefit of this method is that it can be used to compare permeability of various tissues of interest or to serially evaluate changes in P in the same tissue over time. In this study, it was applied to measuring P to albumin as well as to liposomes in granulating and implanted tumor tissues in a rat skin flap window chamber. Changes in permeability to a small molecule (sulforhodamine B) before and during bradykinin application were also measured. The results of these experiments indicate that the relative plasma concentrations predicted by this method conformed well to those measured directly from blood samples, and the measured permeability values were consistent with previously published data. Therefore, this method provides a valid approach for quantitatively measuring the extravasation of intravenously injected molecular and colloidal materials in microcirculatory preparations. The method has a set of defined experimental conditions and assumptions that cannot be violated, however, or erroneous results can be obtained.